Peptide aptamer interactions in a mammalian cell context.

<p>(A) Mammalian cell two-hybrid assay. HeLa cells were co-transfected with a plasmid directing the expression of a GAL4-RasGAP SH3 bait protein, a plasmid containing a luciferase two-hybrid reporter gene, and plasmids directing the expression of VP16-peptide aptamer fusions. Luminescence was measured 48 h after transfection. Two independent experiments were performed. SH3-pACT: RasGAP bait with empty prey plasmid; pBIND-pACT: empty bait and prey plasmids; Control-pACT: empty prey plasmid only. (B) Pull-down assay from total protein extract. Left: A mouse liver total protein extract was added to glutathion sepharose beads, uncoated (−) or coated with GST-RG27 or GST-Control fusion proteins. Captured native RasGAP protein was revealed with a monoclonal antibody directed against the SH3 domain. Right: Coomassie staining of a SDS-PAGE revealing comparable amounts of GST-Control and GST-RG27 fusion proteins immobilized on glutathion sepharose beads.